New way to label nucleic acids could lead to new therapies

Nucleic acids embrace many more kinds of molecules than just giant strands of DNA and RNA, and several forms have shown promise for therapy.

"Nucleic acids and their constituents have been studied for several years as potential therapeutic agents," says Manouchehr Saljoughian of Berkeley Lab's National Tritium Labelling Facility (NTLF). "The most advanced of these are just reaching the clinical trial stage."

Among the types of nucleic acid with promise for therapy are antisense agents -- groups of nucleotides that bind to a strand of messenger RNA and disrupt the passage of genetic information -- and aptamers, sequences of DNA and RNA that bind to a variety of specific molecules with roles in controlling gene expression in diseases such as cancer or atherosclerosis and in other biological functions.

Nucleotides are the structural subunits of DNA and RNA molecules, consisting of sugar-phosphate backbones attached to purine or pyrimidine bases.

Recently Saljoughian and his NTLF colleagues Hiromi Morimoto and Philip G. Williams have found a new way of tagging nucleotides for biochemical studies.

One of the best ways to study how a chemical is distributed and altered in the body is to label it with a trace amount of a radioactive element. Tritium is the radioactive isotope of hydrogen, with two neutrons in its nucleus; since hydrogen is ubiquitous in living tissues, in principle tritium can be used to label an almost limitless range of biological molecules.

In practice, the job is not so easy. For example, nucleotides whose phosphate-backbone segments have been labeled with radioactive phosphorus are commercially available, but the phosphates in these segments can separate from the rest of the nucleotide during metabolism. To track each part of the nucleotide requires additional labeling approaches.

Not only have Saljoughian and his colleagues devised a method for labeling specific bases, but they can place tritium atoms at particular positions on the base or independently at positions on the nucleotide's sugar. An added advantage is that the new phosphorylation technique allows the nucleotides to be tritiated at a very late step of synthesis -- avoiding the need to work with tritiated compounds at earlier stages.

"The phosphorylation method we use was developed to simplify the addition of triphosphates to nucleosides in bulk, without having to worry about unprotected NH and OH groups, which could potentially interfere with the reaction," says Saljoughian. (A nucleoside is a nucleotide without the phosphate.)

Saljoughian explains that they have adapted the method to a "mini-scale" procedure and modified it to synthesize a number of nucleotides, "including particular precursors of adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP), which we can subsequently label with tritium."

The process involves successive stages of drying, dissolving, stirring, adding other reagents and solutions, and drying again -- all in one "pot" -- until the compound is ready to be analyzed for purity.

Saljoughian and Morimoto have also developed a method of monitoring the progress of phosphorylation using high-performance liquid chromatography, particularly useful since the rates of phosphorylation vary greatly between the various nucleosides, and reaction times have to be adjusted accordingly.

In the first steps of the ATP or dATP labeling process, a bromine atom is attached to the base ring where the tritium atom will eventually be added. After a series of other steps, the triphosphate group is attached, and in the labeling step the bromine is removed in the presence of tritium gas and a catalyst, which substitutes tritium atoms at the desired positions in the molecule. The labeled nucleotide is then enzymatically or chemically incorporated into the desired DNA or RNA molecule.

As an example of the research being done with tritiated nucleic acids, Saljoughian cites a collaboration with Scott Taylor of the Lab's Life Sciences Division to synthesize a sequence of the DNA aptamer for thrombin -- a factor essential in blood-clotting -- and attach the tritiated dATP nucleotide to its terminus. Thrombin plays an important role in arterial disease, and binding a specific, selective nucleic acid to a thrombin molecule is one way of putting it out of business.

"The system is now being used to test the specificity of the aptamer for thrombin, as well as the stability of the bound system," Saljoughian says.

Saljoughian explains that during the development of a novel labeling technique, any new procedure is first tested with ordinary hydrogen at a small scale, and if successful, with deuterium (the nonradioactive isotope of hydrogen with one neutron), to make sure that hydrogen does not sneak in from undesired sources. Only then is the process tried with tritium, using only about 10 percent of the tritium required for a compound with high specific activity. Only when the process is judged workable and safe is labeling conducted at full strength.

Part of the National Tritium Labelling Facility's job is to pioneer techniques too difficult or too capital-intensive for universities and industry to handle alone.

In addition to the tritiated nucleotides that Saljoughian and his colleagues have already synthesized, their new method should be useful for synthesizing many related compounds. The prospect of extending fundamental research further into the function and uses of nucleic acids powerfully motivates the efforts of Saljoughian, Morimoto, Williams, Chit Than, principal investigator David Wemmer, and the users and collaborators of the NTLF.